PROJECT SUMMARY/ABSTRACT Approximately 700,000 patients in the United States have a primary central nervous system tumor, and ~200,000 new cases of brain metastases are diagnosed annually. The current approach for brain tumor diagnosis is mainly through neuroimaging [e.g., magnetic resonance imaging (MRI)] to detect tumors with sufficient mass followed by surgical resection or stereotactic biopsy for histologic confirmation of imaging findings. However, tissue biopsies carry significant risk, are not feasible for repeated sampling to monitor the tumor progression and treatment response, and may not be possible at all in surgically inaccessible tumor locations or medically inoperable patients. Blood-based liquid biopsy offers a noninvasive approach to classify disease, guide therapy, monitor treatment response, and unravel molecular mechanisms underlying the disease through the detection of circulating tumor biomarkers (e.g., DNA, RNA, extracellular vesicles, and proteins shed by tumor cells). It has transformed the clinical management of several cancers outside the brain. However, extending blood-based liquid biopsies to brain cancer is challenging mainly because the blood-brain barrier (BBB) hinders the transfer of tumor-derived biomarkers into the blood circulation system. To overcome this challenge, our group introduced the focused ultrasound-enabled brain tumor liquid biopsy (FUS-LBx) technique for noninvasive and spatially targeted molecular characterization of brain tumors. The central hypothesis is that FUS-mediated BBB disruption opens ?two-way trafficking? between brain and blood pool, thereby releasing brain biomarkers into the blood circulation as well as allowing circulating drugs to enter the brain. Our long-term goal is to transform the clinical management of patients with brain cancer by providing molecular signatures of the disease using noninvasive FUS-LBx. The objective of this application is to obtain compelling preclinical evidence needed to support future clinical translation of FUS-LBx. Our objective will be achieved by completing the following three specific aims: (1) Evaluate the impact of FUS parameters on brain-tumor biomarker-release levels and safety in a mouse brain tumor model; (2) Evaluate the impact of tumor variables on FUS-mediated brain-tumor biomarker-release levels in mouse models of brain tumors; (3) Assess the feasibility and safety of FUS-LBx in a pig brain tumor model. The proposed research contains three main innovations: (1) The hypothesis that FUS- induced BBB disruption enables two-way trafficking between blood and brain opens a new research field; (2) FUS-LBx is an innovative diagnostic tool and provides a new pathway to clinical translation of FUS technology; (3) The pig brain tumor model that will be used in this study provides a large animal model that is critical for obtaining unequivocal evidence to support the clinical translation of FUS-LBx. This project is significant because this innovative technique has great potential to radically advance the diagnosis and monitoring of brain cancer patients by identifying molecular signatures of the tumor without surgery.